Arabidopsis camelliol C synthase evolved from enzymes that make pentacycles

Article abstract of DOI:10.1021/ol702399g

(Chemical Equation Presented) We establish by heterologous expression that the Arabidopsis thaliana oxidosqualene cyclase At1g78955 (CAMS1) makes camelliol C (98%), achilleol A (2%), and β-amyrin (0.2%). CAMS1 is the first characterized cyclase that generates predominantly a monocyclic triterpene alcohol. Phylogenese analysis shows that CAMS1 evolved from enzymes that make pentacycles, thus revealing that its pentacyclic β-amyrin byproduct is an evolutionary relic. Sequence alignments support prior suggestions that decreased steric bulk at a key active-site residue promotes monocycle formation.

Full text of DOI:10.1021/ol702399g

ORGANIC
LETTERS
2007
Vol. 9, No. 25
5223-5226
Arabidopsis Camelliol C Synthase
Evolved from Enzymes That Make
Pentacycles
Mariya D. Kolesnikova,† William K. Wilson,‡ David A. Lynch,‡
Allie C. Obermeyer,† and Seiichi P. T. Matsuda*,†,‡
Department of Chemistry and Department of Biochemistry and Cell Biology,
Rice UniVersity, Houston, Texas 77005
matsuda@rice.edu
Received October 2, 2007
ABSTRACT
We establish by heterologous expression that the Arabidopsis thaliana oxidosqualene cyclase At1g78955 (CAMS1) makes camelliol C (98%),
achilleol A (2%), and -amyrin (0.2%). CAMS1 is the first characterized cyclase that generates predominantly a monocyclic triterpene alcohol.
Phylogenetic analysis shows that CAMS1 evolved from enzymes that make pentacycles, thus revealing that its pentacyclic -amyrin byproduct
â
â
is an evolutionary relic. Sequence alignments support prior suggestions that decreased steric bulk at a key active-site residue promotes
monocycle formation.
Triterpene synthases1 guide cationic cyclizations and rear-
rangements to convert squalene or oxidosqualene (1) to
diverse skeletons, which usually contain four or five rings.2
We show here that the Arabidopsis thaliana gene At1g78955
(LUP3)3 encodes an enzyme that converts oxidosqualene to
camelliol C (2) and two minor byproducts. This is the first
example of a native oxidosqualene cyclase that makes an
A-ring monocycle as the dominant product.1a,4 Our phylo-
genetic analysis indicates that this camelliol C synthase
(CAMS1) is an evolutionary descendent of enzymes that
make larger ring systems.
† Department of Chemistry.
‡ Department of Biochemistry and Cell Biology.
(1) Review: (a) Phillips, D. R.; Rasbery, J. M.; Bartel, B.; Matsuda, S.
P. T. Curr. Opin. Plant Biol. 2006, 9, 305-314. Subsequent reports on
triterpene synthases: (b) Tansakul, P.; Shibuya, M.; Kushiro, T.; Ebizuka,
Y. FEBS Lett. 2006, 580, 5143-5149. (c) Sawai, S.; Shindo, T.; Sato, S.;
Kaneko, T.; Tabata, S.; Ayabe, S.-i.; Aoki, T. Plant Sci. 2006, 170, 247-
257. (d) Basyuni, M.; Oku, H.; Inafuku, M.; Baba, S.; Iwasaki, H.; Oshiro,
K.; Okabe, T.; Shibuya, M.; Ebizuka, Y. Phytochemistry 2006, 67, 2517-
2524. (e) Shibuya, M.; Xiang, T.; Katsube, Y.; Otsuka, M.; Zhang, H.;
Ebizuka, Y. J. Am. Chem. Soc. 2007, 129, 1450-1455. (f) Xiang, T.;
Shibuya, M.; Katsube, Y.; Tsutsumi, T.; Otsuka, M.; Zhang, H.; Masuda,
K.; Ebizuka, Y. Org. Lett. 2006, 8, 2835-2838. (g) Kolesnikova, M. D.;
Obermeyer, A. C.; Wilson, W. K.; Lynch, D. A.; Xiong, Q.; Matsuda, S.
P. T. Org. Lett. 2007, 9, 2183-2186. (h) Lodeiro, S.; Xiong, Q.; Wilson,
W. K.; Kolesnikova, M. D.; Onak, C. S.; Matsuda, S. P. T. J. Am. Chem.
Soc. 2007, 129, 11213-11222.
(2) Reviews: (a) Wendt, K. U.; Schulz, G. E.; Corey, E. J.; Liu, D. R.
Angew. Chem., Int. Ed. 2000, 39, 2812-2833. (b) Xu, R.; Fazio, G. C.;
Matsuda, S. P. T. Phytochemistry 2004, 65, 261-290.
(3) Husselstein-Muller, T.; Schaller, H.; Benveniste, P. Plant Mol. Biol.
2001, 45, 75-92.
(4) The only other oxidosqualene cyclase reported to make fewer than
three rings is marneral synthase (MRN1), which constructs a bicyclic cation
that undergoes Grob fragmentation to the seco-A product (technically a
monocycle): Xiong, Q.; Wilson, W. K.; Matsuda, S. P. T. Angew. Chem.,
Int. Ed. 2006, 45, 1285-1288.
10.1021/ol702399g CCC: $37.00
© 2007 American Chemical Society
Published on Web 11/07/2007

CAMS1 was characterized by expression in Saccharomy-
ces cereVisiae (for details, see the Supporting Information).
The confirmed coding sequence was cloned into pRS426GAL5
to generate plasmid pMDK4.8, which was expressed in the
yeast strains SMY86 and RXY6.7 The cell pellet from a 4 L
culture of SMY8[pMDK4.8] was saponified, and the hexane
extract of this in vivo experiment was analyzed by GC-
MS and 1H NMR, both of which displayed dominant signals
consistent with data reported8 for camelliol C (2). Minor
signals corresponding to achilleol A (3)9 and â-amyrin (4)10
were also present. The bulk of the material was purified to
discrete compounds, which were definitively characterized
as 2, 3, and 4 by 800 MHz 1H NMR. To establish the product
ratios, we performed an in vitro reaction with synthetic
oxidosqualene and enzyme present in the RXY6[pMDK4.8]
Table 1. Comparison of Product Accuracy among Cyclasesa
triterpene synthase
P1/P2
P1/∑Pi
reference
LUP3 (CAMS1)
PEN5 (MRN1)
LUP1
PEN1
PEN2 (BARS1)
∼50
>99
0.98
0.99
0.4
this work
4
1a
1g
1h
∼1
20-25
∼33
0.90
a Cyclase accuracy is quantified as the ratio of the primary product
to the second most abundant product (P1/P2) or to total products
(P1/∑Pi).1h
of CAMS1 to generate minor products in the 0.1-1% range
is obviously much lower than that of BARS1 and PEN1.
The high product accuracies of CAMS1 and MRN14 are
partially attributable to their short biosynthetic pathways. For
example, CAMS1 needs only to exclude rearrangement,
further cyclization, and aberrant deprotonation of cation I
(Scheme 1). In contrast, cyclases that make tetracycles and
1
homogenate.11 GC-MS (Figure 1) and H NMR analyses
Scheme 1. Mechanistic Pathway for the Formation of
Camelliol C (2), Achilleol A (3), and â-Amyrin (4) from
Oxidosqualene (1)
Figure 1. GC-MS total ion chromatogram of the underivatized
triterpene fraction from SMY8[pMDK4.8]. Asterisks (*) denote
non-triterpene components, as judged by their mass spectra.
of the in vitro reaction extract confirmed that these com-
1
pounds were enzymatic cyclization products, and H NMR
provided quantitation for the relative amounts of 2 (98%), 3
(2%), and 4 (0.2%). Details of spectral analyses are given
in the Supporting Information.
CAMS1 shows high product specificity, being substantially
more accurate than many Arabidopsis cyclases that function
in secondary metabolism, such as LUP1, BARS1,1h and
PEN11g (Table 1).12 Although the enumeration of trace-level
cyclase products is necessarily incomplete, the propensity
(5) (a) Hart, E. A.; Hua, L.; Darr, L. B.; Wilson, W. K.; Pang, J.; Matsuda,
S. P. T. J. Am. Chem. Soc. 1999, 121, 9887-9888. For details, see: (b)
Hua, L. Ph.D. Thesis, Rice University, 2000, p 76.
(6) SMY8 is a lanosterol synthase deletion mutant that accumulates the
substrate oxidosqualene and consequently supports in vivo biosynthesis:
Corey, E. J.; Matsuda, S. P. T.; Baker, C. H.; Ting, A. Y.; Cheng, H.
Biochem. Biophys. Res. Commun. 1996, 219, 327-331.
(7) RXY6 is a squalene epoxidase/lanosterol synthase double deletion
mutant that is useful for in vitro work because it cannot generate
oxidosqualene: Fazio, G. C.; Xu, R.; Matsuda, S. P. T. J. Am. Chem. Soc.
2004, 126, 5678-5679.
(8) Akihisa, T.; Arai, K.; Kimura, Y.; Koike, K.; Kokke, W. C. M. C.;
Shibata, T.; Nikaido, T. J. Nat. Prod. 1999, 62, 265-268.
(9) Barrero, A. F.; Alvarez-Manzaneda, E. J.; Alvarez-Manzaneda, R.
Tetrahedron Lett. 1989, 30, 3351-3352.
(10) Goad, L. J.; Akihisa, T. Analysis of Sterols; Blackie (Chapman &
Hall): London, 1997; appendix 3, section (h).
pentacycles generate many carbocationic intermediates, each
of which is subject to premature deprotonation or rearrange-
ment.
Phylogenetic analysis shows a close relationship between
CAMS1 and enzymes that generate polycyclic triterpene
alcohols (Figure 2). CAMS1 is most closely related to other
oxidosqualene cyclases in the Arabidopsis LUP clade (70-
78% identical), which form pentacycles or tetracycles. More
distantly related are families of enzymes that make the
(11) We favor in vitro yields because in vivo accumulation can distort
the enzymatic product profile. See: (a) Joubert, B. M.; Hua, L.; Matsuda,
S. P. T. Org. Lett. 2000, 2, 339-341. (b) Lodeiro, S.; Wilson, W. K.; Shan,
H.; Matsuda, S. P. T. Org. Lett. 2006, 8, 439-442.
(12) Several other cyclases have qualitatively high product ratios,1a but
because minor products were not quantitated and detection limits were not
reported, quantitative measures of product specificity are unavailable.
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Org. Lett., Vol. 9, No. 25, 2007

Figure 2. Computed phylogenetic relationships among known1a,c
plant oxidosqualene cyclases closely related to CAMS1. The
nucleotide sequences of these enzymes were aligned with the
MegAlign program (DNASTAR, Inc., Madison, WI) by the Clustal
W method. The alignment was used to create a phylogenetic tree
rooted with the A. thaliana CAS1 using PAUP 4.05b. The tree was
generated using the bootstrap method with 100 replicates with equal
weight given to all the characters and maximum likelihood as the
optimality criterion. Bootstrap values are listed at nodes. Cyclases
are grouped by color according to phylogeny and product structure.
For the full species names, see Figure 3.
Figure 3. Partial amino acid alignment of oxidosqualene
cyclases.1a,c,15 An asterisk (*) designates position 484 in CAMS1,
where steric bulk impacts B-ring formation.
elevated amounts of At1g78955 mRNA in Arabidopsis
inflorescence tissue.16
pentacycles â-amyrin (72-75% identical) and lupeol (59-
61% identical).1a,13 One might imagine that cyclases that
polycyclize oxidosqualene evolved from enzymes that form
smaller ring systems by iterative addition of motifs that favor
additional rings. However, phylogenetic analysis indicates
the reverse evolutionary order, i.e., CAMS1 is a descendent
of enzymes that form pentacyclic triterpenoids.
Monocyclic triterpenoids appear to be distributed sparsely
across the vast diversity of higher plants. Compounds 2 and
3 have been found in a handful of asterids (Camellia
sasanqua,8,17a Camellia japonica,8,17a Achillea odorata,9
Bupleurum spinosum,17b and Santolina elegans17c), two
eurosids (Euphorbia antiquorum17d and Garcinia speciosa17e),
the monocots wheat and rice,17a and the fern Polypodiodes
formosana.17f Because monocyclic and polycyclic triterpenes
have rather different spectral and chromatographic sig-
natures, most monocycles were described as components of
plant oils (usually lacking polycyclic triterpenes) rather than
in surveys of triterpene distribution. We suspect that the
monocyclic triterpenes are more widespread than the litera-
ture suggests.
How often have cyclases that generate A-ring monocycles
evolved? This question can be addressed by considering gene
divergences (Figure 2) in the context of organismal relation-
ships. CAMS1 diverged relatively recently from LUP4 within
the LUP clade. LUP enzymes appear to be unique to malvids
(eurosids II). We conclude that CAMS1 arose within the
malvids and is evolutionarily distinct from the unknown
enzymes that generate monocyclic triterpenes in asterids,
monocots, and ferns.18 The phylogenetic analyses suggest
Sequence alignments show that nearly all plant cyclases
contain valine or isoleucine at the position corresponding
to Ala484 in CAMS1 (Figure 3). Previous mutational
studies11a,14 suggested that decreased steric bulk at this
position (notably mutation to alanine or glycine) promotes
the formation of monocycles. Our CAMS1 results strongly
support this proposal. An uncharacterized cyclase from
Betula platyphylla (OSCBPD)15 also encodes alanine at this
position and may likewise be compromised in B-ring
formation.
The overwhelming dominance of 2 in the product profile
of CAMS1 suggests that camelliol C or a metabolite
thereof provides a competitive advantage that achilleol A
does not replicate. The biological role of enzymes and their
products can often be illuminated by microarray data.
Unfortunately, the initial annotation of the Arabidopsis
genome included At1g78955 (CAMS1) as a fusion with
At1g78950 (LUP4), and these genes were not distinguished
in early microarrays. The limited available nonarray expres-
sion data (Arabidopsis MPSS Plus: Gene Analysis) show
(16) Data available at http://mpss.udel.edu/at/.
(17) (a) Akihisa, T.; Koike, K.; Kimura, Y.; Sashida, N.; Matsumoto,
T.; Ukiya, M.; Nikaido, T. Lipids 1999, 34, 1151-1157. (b) Barrero, A.
F.; Haidour, A.; Munoz-Dorado, M.; Akssira, M.; Sedqui, A.; Mansour, I.
Phytochemistry 1998, 48, 1237-1240. (c) Barrero, A. F.; Alvarez-
Manzaneda, E. J.; Herrador, M. M.; Alvarez-Manzaneda, R.; Quilez, J.;
Chahboun, R.; Linares, P.; Rivas, A. Tetrahedron Lett. 1999, 40, 8273-
8276. (d) Akihisa, T.; Wijeratne, E. M. K.; Tokuda, H.; Enjo, F.; Toriumi,
M.; Kimura, Y.; Koike, K.; Nikaido, T.; Tezuka, Y.; Nishino, H. J. Nat.
Prod. 2002, 65, 158-162. (e) Rukachaisirikul, V.; Pailee, P.; Hiranrat, A.;
Tuchinda, P.; Yoosook, C.; Kasisit, J.; Taylor, W. C.; Reutrakul, V. Planta
Med. 2003, 69, 1141-1146. (f) Arai, Y.; Hirohara, M.; Ageta, H.; Hsu, H.
Y. Tetrahedron Lett. 1992, 33, 1325-1328.
(13) (a) Still more distantly related are cyclases of the Arabidopsis PEN
clade,1a three of which make fewer than four rings. (b) Protein sequence
identities were calculated in MegAlign from pairwise alignments.
(14) Matsuda, S. P. T.; Darr, L. B.; Hart, E. A.; Herrera, J. B. R.;
McCann, K. E.; Meyer, M. M.; Pang, J.; Schepmann, H. G. Org. Lett. 2000,
2, 2261-2263.
(15) Zhang, H.; Shibuya, M.; Yokota, S.; Ebizuka, Y. Biol. Pharm. Bull.
2003, 26, 642-650.
Org. Lett., Vol. 9, No. 25, 2007
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that natural selection can readily modify a cyclase that makes
polycycles to instead deprotonate after A-ring formation.
Frequent evolutionary events of this kind may underlie the
punctate distribution of monocyclic triterpenoids across the
plant kingdom.
and the Herman Frasch Foundation funded this research. The
800 MHz NMR spectra were acquired on instrumentation
purchased by the John S. Dunn, Sr. Gulf Coast Consortium
for Magnetic Resonance.
Supporting Information Available: Experimental pro-
cedures and NMR and GC-MS spectra. This material is
available free of charge via the Internet at http://pubs.acs.org.
Acknowledgment. The National Science Foundation
(MCB-0209769), The Robert A. Welch Foundation (C-1323),
(18) If the Betula platyphylla OSCBPD enzyme15 (noted above) makes
monocycles, this would represent a second evolution of an enzyme that
forms monocycles within the eurosids because CAMS1 is too distant (63%
identical) to be an ortholog.
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